Gene Copy Number and Gene Expression
Normal human cells contain 46 chromosomes in 22 autosome pairs and 2 sex chromosomes. Generally, normal cells contain two copies of every gene (except sex-linked genes in males). In both constitutional genetic diseases such as Down syndrome and acquired genetic diseases such as cancer, this normal pattern can be disrupted. The gene copy number of some genes may be more than two (a “gain” or amplification of gene copy number) or fewer than two. Chromosome number can also be disrupted, with cancer cells in particular showing patterns of gain or loss of whole chromosomes or chromosome arms. The number of copies of a chromosome is also referred to as its “ploidy”.
In cancer, it frequently happens that the copy number of some genes is greater (often much greater) than the copy number of their corresponding chromosomes. This phenomenon is at times referred to as gene amplification or amplification. Various patterns of gene amplification are characteristic of certain cancers and some other conditions and can inform diagnosis, prognosis and/or treatment regimes.
Genes influence the biology of a cell via gene “expression,” which refers to the production of the messenger RNA and thence the protein encoded by the gene. Gene copy number is a static property of a cell established when the cell is created; gene expression is a dynamic property of the cell that may be influenced both by the cell's genome and by external environmental influences such as temperature or therapeutic drugs.
In genetic diseases, gene expression and/or protein expression is also frequently disrupted. In cases where a gene is gained or amplified there is often (though not invariably) a corresponding increase in the expression of that gene, referred to as overexpression. Thus, amplification and overexpression are often, but not always, correlated.
Thus, it is frequently desired to measure and/or determine and/or estimate gene copy number in cells and/or tissues. At present, gene copy number can be measured using a variety of techniques, including quantitative PCR, in situ measuring, and other techniques that attempt to count or estimate the number of specific genetic sequences.
In Situ Hybridization and FISH
The technique of fluorescent in situ hybridization (FISH) is used in a variety of clinical and research settings. Generally, the technique is used to locate chromosomal location(s) of specific DNA (or RNA) sequences. A complementary probe is labeled with a fluorescent dye and is then added to a chromosomal or cell preparation from the species of interest. After a sufficient time for annealing to occur, the chromosomes are viewed using a fluorescent microscope. The probe will hybridize to the chromosome carrying the sequence of interest. If the sequence has been characterized cytogenetically, the marker can be assigned to the appropriate chromosome.
FISH analysis has been useful for studying human diseases. For example, if a patient suffering a disease is determined via FISH analysis to have a deletion at a specific chromosomal locus, then the gene responsible for the disease is likely to reside on the missing segment. FISH analysis of tumor tissues can in some cases reveal chromosomal additions, deletions and/or substitutions that may be characteristic of some cancers or other conditions of interest.
More recently, many various strategies and techniques have been proposed for improving and/or automating research and/or diagnostic tests using FISH analysis. Many references describe a range of techniques and methods utilizing FISH. Among these are the following issued U.S. Pat. Nos. 4,833,332; 5,780,857; 5,830,645; 5,936,731; 6,146,593; 6,210,878; 6,225,636; and 6,242,184.
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